Peroxynitrite-dependent formation of nitrotyrosine has been associated with inactivation of various enzymes and proteins possessing functionally important tyrosines. We have previously reported an enzymatic activity modifying the nitrotyrosine residues in nitrated proteins. Here we are describing a nonenzymatic reduction of nitrotyrosine to aminotyrosine, which depends on heme and thiols. Various heme-containing proteins can mediate the reaction, although the reaction also is catalyzed by heme. The reaction is most effective when vicinal thiols are used as reducing agents, although ascorbic acid also can replace thiols with lesser efficiency. The reaction could be inhibited by (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1, but not other tested NO donors. HPLC with electrochemical detection analysis of the reaction identified aminotyrosine as the only reaction product. The reduction of nitrotyrosine was most effective at a pH close to physiological and was markedly decreased in acidic conditions. Various nitrophenol compounds also were modified in this reaction. Understanding the mechanism of this reaction could help define the enzymatic modification of nitrotyrosine-containing proteins. Furthermore, this also could assist in understanding the role of nitrotyrosine formation and reversal in the regulation of various proteins containing nitrotyrosine. It also could help define the role of nitric oxide and other reactive species in various disease states.aminotyrosine ͉ nitric oxide N itric oxide (NO) is a signaling molecule generated from L-arginine via the catalytic action of both constitutive and inducible forms of NO synthases (1). A large body of evidence has been accumulated in the last decade establishing the role of NO in the pathogenesis of inflammatory, infectious, and degenerative human disease (2). The detrimental effects ascribed to NO often arise from its conversion to more reactive species through reactions with partially reduced oxygen (3).Recent evidence supports a role for peroxynitrite in cellular damage and apoptosis (4-8) that was previously attributed entirely to NO. The formation of peroxynitrite from nitric oxide and superoxide anion is almost diffusion-limited (9). Peroxynitrite has been shown to cause lipid peroxidation (10-12), chemical cleavage of DNA (7,13,14), inactivation of key metabolic enzymes such as aconitase (15), ribonucleotide reductase, succinate dehydrogenase, and cytochrome oxidase of the mitochondrial electron transport chain (16)(17)(18)(19). Peroxynitrite can also nitrate protein tyrosine residues, leading to decreased tyrosine phosphorylation by tyrosine kinases (20,21) or to alteration of the functions of proteins͞enzymes in vitro (22, 23) and perhaps in vivo (24,25). It has been reported that nitrotyrosine formation in cytoskeletal components can alter their function (26). The present view is that protein nitration is associated with some toxic or deleterious effects of nitric oxide and with the interruption of cellular signaling processes. The format...